Functionalized Cortical Bone‐Inspired Composites Adapt to the Mechanical and Biological Properties of the Edentulous Area to Resist Fretting Wear

Abstract Dental implants with long‐term success of osseointegration have always been the goal, however, difficulties exist. The accumulation of fretting damage at the implant–bone interface often gets overlooked. Commonly used titanium is approximately 7‐fold harder and stiffer than cortical bone. Stress shielding caused by the mismatching of the elastic modulus aggravates fretting at the interface, which is accompanied by the risk of the formation of proinflammatory metal debris and implant loosening. Thus, the authors explore functionalized cortical bone‐inspired composites (FCBIC) with a hierarchical structure at multiple scales, that exhibit good mechanical and biological adaptivity with cortical bone. The design is inspired by nature, combining brittle minerals with organic molecules to maintain machinability, which helps to acquire excellent energy‐dissipating capability. It therefore has the comparable hardness and elastic modulus, strength, and elastic‐plastic deformation to cortical bone. Meanwhile, this cortical bone analogy exhibits excellent osteoinduction and osseointegration abilities. These two properties also facilitate each other to resist fretting wear, and therefore improve the success rate of implantation. Based on these results, the biological–mechanical co‐operation coefficient is proposed to describe the coupling between these two factors for designing the optimized dental implants.

Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were conducted using a Thermogravimetric analyzer (Perkin-Elmer 7, USA) under a nitrogen atmosphere with a heating rate of 10 ℃ min -1 . Fourier transform infrared spectroscopy (FTIR) was performed using an infrared spectrometer (Boguang Technology Co. Ltd., Shanghai, China). The elemental groups on the surface of the material before and after functionalization were characterized using X-ray photoelectron spectroscopy (XPS, AXIS Supra, Kratos, US). Fresh bovine cortical bones in edentulous area were obtained to donated by patients who had signed an informed consent form. The FCBIC slices were cut through a focused ion beam (FIB, FEI Strata 400S, USA) and a high-angle annular dark field (HAADF) transmission electron microscopy mode (STEM) image of the sliced specimens was obtained using TEM.

Mechanical properties
Rheological experiments on 53 wt% nano-3Y-TZP and pDA-3Y-TZP slurry were performed using a high-shear rheometer (Discovery HR-20, TA instruments, USA) with 10 rad/s angular frequency at 25 ℃. Compression and tensile tests were performed using an Instron testing machine (Instron 3400; Instron, MA, US) at room temperature. Cylindrical samples were prepared for monotonic compression tests with a displacement rate of 0.5 mm min -1 . The cyclic compression tests were conducted with a fixed strain rate (10 −3 s −1 ) and a constant displacement increase, by loading and unloading samples repeatedly until the samples failed. [1] Young's moduli and hardness were measured using an indentation test using the TriboIndenter system (Hysitron Inc. TI950, Minneapolis, MN, USA) with a load of 6,000 μN and a trapezoid function including a linear loading period, a hold period at the peak load, and an unloading period for 20 s each on polished samples up to 1 μm . The flexural   3   strength was measured by a three-point bending test using an electro force 3310 series   II test instruments (3310-AT series II, TA instruments, USA). The specimens were 3.0 mm in thickness and 4.0 mm in width. The support span was 20 mm and the bending displacement rate was 0.000083 mm s -1 . The flexural strength was calculated using the following equation:

( )
Where P is the breaking load, l is the length of support span, w is the specimen width, and b is the specimen thickness.

In vitro wear tests
Bovine cortical bones were used because their physical and mechanical properties are similar to that of the human cortical bone. [2] The fretting wear tests were conducted by a fretting and wear tester (FFT-MI, RETC Instrument, USA) with an amplitudes of 100 μm and a tribometer driven by a high-precision piezoelectric ceramic actuator The wear scars were observed through a white light interferometer 3D surface profilometer (RTEC, USA). The frequency of the reciprocating motion of horizontal sliding was 2 Hz, the vertical load was 90 N and 20 N respectively, and 20,000 cycles. [3]

Single-edge notched beam (SENB) testing
Single-edge-bend specimens (width ca. 4.0 mm, thickness ca. 2.0 mm) containing a U-groove notch (depth ca. 1.8 mm) and a V-notch (depth ca. 0.2 mm), in which the root radius for a straight-through slot terminating is about 0.1mm, were tested on an electro force 3310 series II test instrument (3310-AT series II, TA instruments, USA) at a constant displacement rate of 0.000083 mm s -1 with a support span of 16 mm. The plane strain fracture toughness, J IC , which presents the crack-extension resistance under conditions of crack-tip plane-strain, was calculated by J-R curve, which was acquired by quantifying the J-integral as a function of crack extension monitored in situ in the SEM. The J-integral, which characterizes the local stress-strain field around the crack front, can be obtained for pure bending as follows:

( )
Where A is the area under the force versus displacement record, B is the specimen thickness, and b 0 is the distance from the V-notch front to the back edge of the specimen.
The fracture toughness based on stress intensity factor, K, can be calculated using the following equation: where E is the Young's modulus and ν is the Poisson's ratio.

Surface properties
The water contact angle (WCA) was measured by a Contact Angle System (Drop Shape Analyzer-DSA100, KRÜSS, Hamburg, Germany) to evaluate the hydrophilicity of the specimens. The surface roughness was measured using a white 5 light interferometer 3D surface profilometer (RTEC instruments, USA) and the Gwyddion 2.30 opensource scanning probe microscopy analysis software (Czech Metrology Institute, Brno, Czech Republic).

Cell preparation and culture
The hWJ-MSCs were isolated from fresh human umbilical cords donated by patients

Cell morphology
The morphology of hWJ-MSCs for each group was observed by SEM (JSM-IT500, JEOL, Japan) after the cells were cultured in 48-well plates up to 24 h. The cells were gradient dehydrated with ethanol and air-dried, and then metalized with gold.

Immunofluorescence staining
The

Histology and histomorphometry
The specimens were gradient dehydrated with ethanol after μCT analysis. Next, the specimens were embedded in a methyl methacrylate solution at 50 °C until polymerization, after being emersed in dipping solutions I, II, and III for 5 days each.
After, a microtome (LeicaMicrotome, Germany) was used to cut thin sections (ca. 100 μm in thickness) along the horizontal plane of the implant. The thin sections were polished to 20-30 μm in thickness. The sections were stained with methylene blue-acid magenta. The red color represented the mineralized bone tissue. [6] Semi-quantitative analyses were conducted by calculating the percentages of positively-stained areas versus the total field areas. Photoshop software was used to measure and calculate static histomorphometry. Van Gieson's picrofuchsin was used 9 to distinguish the collagen and other tissues connected with implants. [7] The red color represented the collagen fibers. [8]

Statistical analysis
Numerical data are presented as the mean and standard deviation (mean ± SD). The SPSS 24.0 software (SPSS Inc., Chicago, USA) was used to analyze the differences among the groups using a one-way or two-way ANOVA. A P value < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 5.01 (GraphPad Software, La Jolla, CA).